Many have tried to design a lateral flow assay for determining the presence and quantity of analytes in biological samples, such as blood samples that contain whole blood, red blood cells, or white blood cells, but have failed. The reasons for failure are many but may be attributable primarily to factors such as hemolysis of the red blood cells creating high background noise, low filtering efficiency, for example, resulting in leakage of the red blood cells onto the chromatographic strip, requirement for a relatively large sample volume (such as requiring 100 μl of sample or more), low efficiency in dissolving a conjugate or detectable agent, volume variation because of variation in cell volume when cells are present and long assay time. It would be desirable to design a lateral flow assay and system that can overcome one or more of these problems in the prior art.
In addition, it would be desirable to simplify the structure of the test strips for lateral flow assays to improve efficiency of the assay and to reduce manufacturing cost.
U.S. Pat. No. 6,136,610 to Polito et al. describes a method and apparatus for performing a lateral flow assay. U.S. Pat. No. 6,528,323 to Thayer et al. describes a bidirectional lateral flow test strip and method for conducting a lateral flow assay. While the methods and system described in these patents are useful for detecting and quantifying most analytes, these patents do not teach how the methods and system can be used to analyze samples containing cells, including red blood cells and/or white blood cells or other cell types. PCT Published Patent Application No. WO 03/008933 describes a test strip for conducting a lateral flow assay for a sample containing whole cells. However, the test strip in WO 03/008933 can be improved to simplify the structure, improve efficiency, reliability, reduce volume dependency and reduce manufacturing cost.
Different strategies have been applied to remove cells, such as red blood cells, from samples, such as blood samples, for detection of analytes, such as infectious disease organisms or antibodies to the infectious disease organisms. However, until now, few strategies have worked well. For example, U.S. Pat. No. 5,766,552 to Doshi et al. discloses the use of a porous material such as an absorbent pad which contains a mixture of both free agglutinating agents and particle-associated agglutinating agents intimately associated with nucleating particles. This filtering system requires about 100 μl of whole blood as shown in FIG. 4 therein.
Human erythrocytes contain on their cell surfaces several transelement proteins that may be suitable targets for making antibodies to red blood cells. For example, Band 3 is associated with the electroneutral exchange of chloride and bicarbonate across the cell element. Band 3 is a 911 amino acid glycoprotein having a 43 kDa amino-terminal cytosolic domain that binds the cytoskeleton, hemoglobin and glycolytic enzymes, and a 52 kDa carboxyl-terminal element domain that mediates anion transport, as described in Wang, D. N. (1994).
Two peptides of Band 3 have been purified, C1 containing Ala893-Val911 and KS4 containing Gly647-Arg656, as described in Fu, G. et al. (2004). The C1 peptide was found to contain protease activity, cleaving glycophorin A (GPA) at Leu118-Ser 19 in a dose-dependent manner, but the KS4 peptide did not cleave GPA under the same conditions.
Human erythrocytes further contain on their cell surface another protein, glycophorin. Glycophorin A (GPA) has been reported to enhance the expression of Band 3 anion transport activity at the cell surface of Xenopus oocytes. Young, M. T. and Tanner, M. J. (2003). The authors found that the C-terminal cytoplasmic tail of GPA enhanced trafficking of Band 3 to the cell surface, whereas the extracellular residues 68-70 increased the specific anion transport activity of Band 3.
Up to the present, there is lacking a rapid, effective and efficient quantitative lateral flow assay and system that can be used for determination of analytes in biological samples, such as in a blood sample, in a point-of-care setting, or a lateral flow assay and system that can be used for determination of analytes that are present in a small volume of sample, such as from a finger prick, or a lateral flow assay and system that can be used for determination of analytes that is volume independent, or that would address other problems in the prior art lateral flow assays and systems.